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The MEP Pathway in Babesia Orientalis Apicoplast, a Potential

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The MEP Pathway in Babesia Orientalis Apicoplast, a Potential He et al. Parasites & Vectors (2018) 11:452 https://doi.org/10.1186/s13071-018-3038-7 RESEARCH Open Access The MEP pathway in Babesia orientalis apicoplast, a potential target for anti- babesiosis drug development Lan He1,2,3, Pei He1,2, Xiaoying Luo1,2, Muxiao Li1,2, Long Yu1,2, Jiaying Guo1,2, Xueyan Zhan1,2, Guan Zhu4 and Junlong Zhao1,2,3* Abstract Background: The apicomplexan parasite Babesia orientalis, the causative agent of water buffalo babesiosis in China, is widespread in central and south China, resulting in a huge economic loss annually. Currently, there is no effective vaccine or drug against this disease. Babesia bovis and Plasmodium falciparum were reported to possess an apicoplast which contains the methylerythritol phosphate (MEP) pathway inhibitable by fosmidomycin, suggesting that the pathway could serve as a drug target for screening new drugs. However, it remains unknown in B. orientalis. Methods: Primers were designed according to the seven MEP pathway genes of Babesia microti and Babesia bovis.The genes were cloned, sequenced and analyzed. The open reading frames (ORFs) of the first two enzyme genes, 1-deoxy- D-xylulose 5-phosphate synthase (BoDXS) and 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (BoDXR), were cloned into the pET-32a expression vector and expressed as a Trx-tag fusion protein. Rabbit anti-rBoDXS and rabbit anti-rBoDXR antibodies were generated. Western blot was performed to identify the native proteins of BoDXS and BoDXR in B. orientalis. Fosmidomycin and geranylgeraniol were used for inhibition assay and rescue assay, respectively, in the in vitro cultivation of B. orientalis. Results: The seven enzyme genes of the B. orientalis MEP pathway (DXS, DXR, IspD, IspE, IspF, IspG and IspH) were cloned and sequenced, with a full length of 2094, 1554, 1344, 1521, 654, 1932 and 1056 bp, respectively. BoDXS and BoDXR were expressed as Trx-tag fusion proteins, with a size of 95 and 67 kDa, respectively. Western blot identified a 77 kDa band for the native BoDXS and a 49 kDa band for the native BoDXR. The drug assay results showed that fosmidomycin could inhibit the growth of B. orientalis, and geranylgeraniol could reverse the effect of fosmidomycin. Conclusions: Babesia orientalis has the isoprenoid biosynthesis pathway, which could be a potential drug target for controlling and curing babesiosis. Considering the high price and instability of fosmidomycin, further studies should focus on the screening of stable and cheap drugs. Keywords: Babesia orientalis, Apicoplast, Isoprenoid, Methylerythritol phosphate (MEP) pathway, 1-deoxy-D- xylulose 5-phosphate reductoisomerase (DXR), Fosmidomycin * Correspondence: [email protected] 1State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, People’s Republic of China 2Key Laboratory for Development of Veterinary Diagnostic Products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. He et al. Parasites & Vectors (2018) 11:452 Page 2 of 8 Background Babesia spp. are tick-borne intraerythrocytic proto- Many members of the Apicomplexa, including Plasmo- zoans and cause babesiosis globally. Babesia orientalis,a dium, Toxoplasma, Eimeria, Babesia and Theileria, con- recently identified species epidemic in China, causes tain a unique, relic, non-photosynthetic plastid-like water buffalo babesiosis. The parasite is transovarially organelle termed apicoplast [1]. The apicoplast is a transmitted by Rhipicephalus haemaphysaloides which is semi-autonomous organelle which normally contains a the only reported vector of B. orientalis, and water buf- 35 kbp circular genome. There are three pathways in the falo is the only reported host. As a member of the family apicoplast of Plasmodium and Toxoplasma, including Babesiidae, B. orientalis has a similar life-cycle with the methylerythritol phosphate (MEP) pathway for other babesiid species, including a sexual stage within R. isoprenoid biosynthesis, FAS pathway and heme operate. haemaphysaloides, followed by an asexual stage in water In B. bovis and Babesia microti, the isoprenoid biosyn- buffalo RBC. Babesiosis has become one of the most im- thesis MEP pathway is the only metabolic pathway in portant water buffalo diseases in central and south China the apicoplast [2, 3]. Isopentenyl diphosphate (IPP) and [11]. However, there is no commercial vaccine or drug dimethylallyl diphosphate (DMAPP) are two precursors available to cure the disease. While the MEP pathway to construct all the isoprenoids which are important for plays a critical role in the parasites and is reported to be a organisms. Isoprenoids comprise a large group of diverse potential drug target, little information is available on the intracellular metabolites with multiple cellular functions, MEP pathway in B. orientalis.Inthisstudy,weproved including roles in membrane structure, cellular respir- that B. orientalis has the MEP pathway, and fosmidomycin ation and cell signaling [4]. can inhibit the growth of B. orientalis. The results demon- In nature, there are two distinct biosynthetic routes to strate that the MEP pathway could serve as a potential produce IPP and DMAPP: methylerythritol phosphate target for screening anti-babesiosis drugs. (MEP) pathway and a modified mevalonate (MVA) path- way. The mevalonic acid (MVA) pathway was thought to Methods be the only IPP and DMAPP biosynthetic pathway for Parasites decades [5]. Mammals, including humans, use the MVA Babesia orientalis (Wuhan strain) was previously isolated pathway that initiates from mevalonate, whilst eubac- from Wuhan city, Hubei Province, China, and preserved in teria, cyanobacteria, plant chloroplasts and apicom- liquid nitrogen in the State Key Laboratory of Agricultural plexan parasites exploit the methylerythritol phosphate Microbiology, Huazhong Agricultural University, China. (MEP) pathway that starts from pyruvate [6]. Three one-year-old water buffaloes were purchased The MEP pathway is essential in eubacteria and proto- from a Babesia-free area. The buffaloes were tested by zoan but absent in humans and animals which could be the Giemsa staining and loop-mediated isothermal amplifi- hosts of eubacteria and protozoan. The MEP pathway was cation (LAMP) to confirm that they were Babesia free/ discovered in the 1990s [7]. In this pathway, the reaction of negative [12]. One water buffalo was used as a negative all the steps are catalyzed by seven different enzymes, in- control. The other two water buffaloes were splenecto- cluding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), mized 2 weeks before infection, and subcutaneously 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR injected with 4 ml of B. orientalis positive blood (1% or IspC), 4-diphosphocytidyl-2C-methyl-D-erythritol cyti- parasitemia). Blood was collected when the parasitemia dylyltransferase (IspD), 4-diphosphocytidyl-2C-methyl-D-er of the infected buffaloes reached 3%. ythritol kinase (IspE), 2C-methyl-D-erythritol-2, 4-cyclodip hosphate synthase (IspF), 1-hydroxy-2-methyl-2-(E)-buteny DNA extraction l-4-diphosphate synthase (IspG) and 1-hydroxy-2-methyl- Blood from the jugular vein of experimentally infected 2-(E)-butenyl-4-diphosphate reductase (IspH) [6]. The sec- water buffaloes was collected in tubes containing EDTA ond enzyme DXR catalyzes step II of the conversion of (BD Vacutainer, USA). Genomic DNA was extracted 1-deoxy-D-xylulose 5-phosphate (DOXP) to 2-C-methyl-- from 200 μlofB. orientalis-infected blood using the D-erythritol 4-phosphate (MEP), which is a target for QIAamp DNA Mini Kit (Qiagen, Shanghai, China) accord- fosmidomycin [8]. Fosmidomycin, a broad-spectrum anti- ing to the manufacturer’s instructions. The DNA samples microbial agent showing the clinical promise of antimalarial were used immediately or stored at -20 °C until use. drug, inhibits the isoprenoid biosynthesis by suppressing the activity of P. falciparum DXR, a rate-limiting enzyme of RNA extraction and cDNA synthesis the MEP pathway, and the parasites will die for lack of iso- Total RNA was extracted from the purified B. orientalis prenoids (by blocking the conversion of DOXP to merozoites by using the TRIZOL reagent (Invitrogen, 2-C-methyl-D-erythritol 4-phosphate). Fosmidomycin is Shanghai, China) and treated with RNase-free DNaseI currently under Phase II clinical trials in combination with (TaKaRa, Dalian, China). RNA concentration was mea- clindamycin and piperaquine against malaria [8–10]. sured by Nanodrop 2000. The cDNA was prepared from He et al. Parasites & Vectors (2018) 11:452 Page 3 of 8 1 μg of the total RNA using PrimeScriptTM RT reagent per lane) were separated by 12% SDS-PAGE, transferred Kit with gDNA eraser (TaKaRa, Dalian, China) accord- onto nitrocellulose membrane (Millipore, Shanghai,
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